Refinement of the Coomassie Blue Method of Protein Quantitation

Abstract

The Coomassie brilliant blue Cl assay for proteins described by Bradford (1976) (Ana!. Biochem. 72, 248) was reexamined. It was found that the extinction coef-ficient of the dye-protein complex solution remained constant over the protein concentration range of 0.8 to 10 &ml of solution. This unchanging extinction coefficient, A,,, = 0.60 k O.Ol/lO pg of protein/ml of solution, enhances both the sensitivity and versatility of the assay. Selection of a volume of dye-reagent (0.5 to 5.0 ml) which dilutes the protein sample to a final concentration of 0.8 to 10 pg/ml permits the application of Beer's Law for accurate determinations of 50.5 to 50 pg of protein. A combination of Bradford's study and the present one indicates that most common laboratory reagents and chemicals exert little or no influence on the Asg5 of the dye-reagent. Bradford (1) recently described a spectrophotometric assay for proteins that is fourfold more sensitive than the standard assay of Lowry et al. (2). This new assay requires a single reagent, Coomassie brilliant blue G, and only 2 min for development. Since the dye-reagent is relatively un-affected by many common laboratory reagents and biochemicals, it is also considerably more convenient to use. However, when Bradford attempted to increase the sensitivity of the assay by decreasing the volume of the dye-reagent, an unexpected de-crease in the extinction coefficient of the dye-protein complex was noted. The study presented here describes a reinvestigation of the apparently anomalous relationship between the extinction coefficient of the complex and the volume of the dye-reagent. It was found that, within the pro-tein concentration range of 0.8 to 10 &ml, the extinction coefficient of the dye-protein complex did not change when the volume of the dye-reagent was varied from 0.5 to 5 ml. This feature permits the sensitivity of the assay to increase inversely with the volume of the dye-reagent added. 0003-2697/78/0861-0142$02.00/O